The major protein that precipitates in the amyloid deposits in brains of humans and other mammals in aging, in Alzheimer's disease (AD) and Down's syndrome is a 39-43 amino acid protein termed AB. AB results from the proteolytic degradation of a larger precursor, the amyloid precursor protein (APP), and mutations in the APP in the region of AB are associated with familial AD. A second protein invariably associated with AB in amyloid deposits is the serine protease inhibitor alpha 1-antichymotrypsin (ACT). The precipitation of AB may result from the disturbance of the delicate balance between proteases and their inhibitors in brain, and agents that can modulate the proteolytic/inhibitory activities become candidates for therapeutic interventions aimed at lowering the levels of AB. For the past years, the investigators have studied the proteases involved in cleaving APP at or near the N-terminus of AB, and the role of ACT in this process. Their long-term objectives are to purify, clone and characterize the proteases identified in the first two years of this grant, and to prove their relevance to APP degradation. There are four specific aims: The first aim is to complete characterization and cloning of the calcium-activated serine protease (CASP) and compare its levels as a function of age and in AD and age-matched controls. CASP cleaves between lysine and methionine in a synthetic peptide flanking the N-terminus of AB, and can generate amyloidogenic fragments from APP; APP metabolism will be compared in CASP anti sense-transfected and untransfected cells. The second aim is to analyze the novel serine protease cloned from a human brain expression library. The cDNA probe will be used to determine its tissue-specific distribution. Antibodies have been generated against two peptides derived from the cDNA sequence which now will be used in the purification of the protease from tissues and cultured cells. Once purified, the protease activity will be tested against APP. They will also use anti sense RNA to knock-out its activity in primary cells and cell lines and follow the APP degradation in those cells. The third aim is to prove the relevance of a zinc metalloprotease, purified and sequenced by the investigators to APP processing. They will clone the cDNA of this protease and then also use anti sense probes to inactivate it in cultured cells. The fourth aim is to continue studies on ACT-like and cathepsin G-like proteins in primary mouse astrocytes and in human glioblastoma cell lines. In summary, the applicants propose to complete the characterization of three serine proteases and one metalloprotease and to prove their relevance to APP degradation and AD development.